IGEM:Brown/2007/Lab Protocols/Pouring Plates 2

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Pouring plates

1. Prepare the media

Prepare 1 liter of LB agar. Use a 2 liter flask and add 1 l of distilled water from the DW tap at the sink. Add 32 g of LB agar. LB agar is kept on the shelves in the sequencing aisle. Swirl to dissolve. Cover the flask with aluminum foil (on top of small fridge close to the door) and use autoclave tape to mark the foil.

2. Autoclave

Run a 15 min liquid cycle.

3. Cool media to 50 C

Put the media in the 65C water bath next to the window till it is cool enough to hold. Make sure to mix well the media. There can be some density separation during autoclaving so that the top is more aqueous (which will make runny plates) and the bottom is more agar like (making plates which set up really quickly).

4. Add the antibiotic

For Ampicillin plates, add ampicillin to a concentration of 50 ug / ml. The ampicillin is mixed up ahead in water and filter sterilized. For 4 mg/ml AMP add 12.5 ml. For 100 mg/ml AMP add 0.5 ml.

5. Pour the plates

Plates are kept at the end of the lab near the sink. We typically use the 4" plates, not the great big ones. Wipe down a lab bench. Pour plates to a depth of 4 or 5 mm and cover. Typically you get about 40 plates per 1 l batch. Let plates set until cool.

6. Store the plates in refridgerator.

Stack the plates back into the original bags. Store the plates upside down in the fridge or cold room until needed.

KC 3/02